Method of detecting drug-receptor and protein-protein interactions

ABSTRACT

The present invention relates to methods for detecting interactions between two proteins as well as detecting the modulation of those interactions. The present invention is based upon the discovery of a new non-nuclear system utilizing G protein gamma subunit fusions to detect the interactions between two proteins and is particularly useful for the detection of the interaction between two or more proteins wherein one of the proteins is associated with the cell membrane. Related methods, compositions and kits can be used to detect or assay the interactions between essentially any two proteins that can be expressed in a cell.

CROSS-REFERENCE TO RELATED APPLICATION

This application claims the benefit of U.S. Provisional Application60/082,940, filed Apr. 24, 1998, which is herein incorporated byreference in its entirety.

ACKNOWLEDGMENT OF FEDERAL SUPPORT

The present invention arose in part from research funded by thefollowing federal grant monies: GM 553160.

TECHNICAL FIELD

The present invention relates to methods for detecting interactionsbetween two proteins as well as detecting the modulation of thoseinteractions. The present invention is based upon the discovery of a newnon-nuclear system to detect the interactions between two proteins andis particularly useful for the detection of the interaction between twoor more proteins wherein one of the proteins is associated with the cellmembrane. Related methods, compositions and kits can be used to detector assay the interactions between essentially any two proteins that canbe expressed in a cell.

BACKGROUND OF THE INVENTION

A fundamental area of inquiry in biology is the analysis of interactionsbetween proteins. Proteins are complex macromolecules made up ofcovalently linked chains of amino acids. Each protein assumes a uniquethree dimensional shape determined principally by its sequence of aminoacids. Many proteins consist of smaller units termed domains, which arecontinuous stretches of amino acids able to fold independently from therest of the protein. Some of the important forms of proteins areenzymes, polypeptide hormones, receptors, nutrient transporters,structural components of the cell, hemoglobins, antibodies,nucleoproteins, and components of viruses.

Protein-protein interactions enable two or more proteins to associate. Alarge number of non-covalent bonds form between the proteins when twoprotein surfaces are precisely matched. These bonds account for thespecificity of recognition. Protein-protein interactions are involved,for example, in the assembly of enzyme subunits; in antigen-antibodyreactions; in forming the supramolecular structures of ribosomes,filaments, and viruses; in transport; and in the interaction ofreceptors on a cell with growth factors and hormones. Products ofoncogenes can give rise to neoplastic transformation throughprotein-protein interactions. For example, some oncogenes encode proteinkinases whose enzymatic activity on cellular target proteins leads tothe cancerous state. Other examples of protein-protein interactioninclude when a virus infects a cell by recognizing a polypeptidereceptor on the surface and when platelets aggregate during thrombosis.

Protein-protein interactions have been generally studied in the pastusing biochemical techniques such as cross-linking,co-immunoprecipitation and co-fractionation by chromatography. Adisadvantage of these techniques is that interacting proteins oftenexist in very low abundance and are, therefore, difficult to detect.Another major disadvantage is that these biochemical techniques involveonly the proteins, not the genes encoding them. When an interaction isdetected using biochemical methods, the newly identified protein oftenmust be painstakingly isolated and then sequenced to enable the geneencoding it to be obtained. Another disadvantage is that these methodsdo not immediately provide information about which domains of theinteracting proteins are involved in the interaction.

To alleviate the problems associated with the biochemicalcharacterization of protein-protein interactions, genetic systems havebeen invented that are capable of rapidly detecting which proteinsinteract with a known protein, determining which domains of the proteinsinteract, and providing the genes for the newly identified interactingproteins. One such system is the yeast two-hybrid system wherein twoproteins are expressed in yeast: one protein of interest fused to aDNA-binding domain and the other protein of interest fused to atranscriptional activation domain (Fields et al. (1989) Nature 340:245;Gyuris et al. (1993) Cell 75:791; Harper et al. (1993) Cell 75:805;Serrano et al. (1993) Nature 366:704; and Hannon et al. (1993) Genes &Dev. 7:2378). If the proteins interact, they activate transcription of areporter gene that contains a binding site for the DNA-binding protein.

Although the development of genetic systems that utilize directactivation of a reporter gene, such as the yeast two-hybrid systems, hasgreatly facilitated the study of protein-protein interactions, manyproblems remain to be solved. For instance, the yeast two-hybrid systemsrely on interactions between the two proteins in the nucleus of thecell. Accordingly, yeast two-hybrid systems are not useful for the studyof integral membrane protein interactions and cannot be used to testcell membrane impermeate drugs. Furthermore, the study ofprotein-protein interactions wherein one of the proteins is itself atranscriptional activator often results in the transcription of thereporter gene without interaction between the two proteins under study.Lastly, the yeast two-hybrid systems require that both proteins understudy be expressed as fusion proteins resulting in the possible loss offunction.

SUMMARY OF THE INVENTION

The present inventors have discovered a genetic system to studyprotein-protein interactions that solves many of the problems associatedwith existing genetic systems. This system utilizes the receptor-Gprotein signaling system present in yeast and other eukaryotic cells tostudy protein-protein interactions, including the interactions whereinone protein is an integral membrane protein.

The present invention includes kits, vectors and methods for detectingone or more interactions between two proteins, comprising the steps ofproviding a cell with a first protein fused to a G protein gamma subunitand a second protein; and determining whether the interaction of the Gprotein gamma subunit with an effector molecule has been modulated,thereby determining whether the first and second proteins interact.

The present invention also includes kits, vectors and methods toidentify an agent that modulates at least one interaction between twoproteins, comprising the steps of exposing a cell to the agent, the cellcomprising a first protein fused to a G protein gamma subunit and asecond protein; and determining whether the interaction of the G proteingamma subunit with an effector molecule has been modulated.

The present invention further provides kits, vectors and methods toidentify binding partners of a protein, comprising the steps oftransforming or transfecting host cells with a library comprising apopulation of nucleic acid molecules which express a first protein fusedto a G protein gamma subunit to produce a host cell population, saidpopulation of nucleic acids differing with respect to the first proteinfused to a G protein gamma subunit; transforming the host cellpopulation with a vector which expresses a second protein; culturingsaid host cell under conditions which express said first and secondprotein; and determining the activity of an effector molecule which iscapable of interacting with the G protein gamma subunit.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B.

FIGS. 1A and 1B are schematics comparing the traditional yeasttwo-hybrid methods of detecting protein-protein interactions to themethods of the invention. Proteins X and Y of FIG. 1B correspond to thefirst and second proteins of the claimed methods, respectively.

FIGS. 2A-2B.

FIGS. 2A and 2B are schematics of pRS314-GAL-rbSec1-H6-STE18 andpRS316-ADH-syntaxin.

FIGS. 3A-3B.

FIG. 3A represents the growth inhibition results of an assay to detectthe ability of Sec-1 and syntaxin to interact and prevent pheromoneinduced growth inhibition.

FIG. 3B is a schematic of the elements used in each growth inhibitionassay.

MODES OF CARRYING OUT THE INVENTION General Description

The present invention utilizes the heterotrimeric G protein (guaninenucleotide binding regulatory protein) regulatory mechanisms found inall eukaryotic cells to study protein-protein interactions. Whenfunctioning normally, G proteins act as an integral part of the signaltransduction mechanisms by which extracellular hormones and otherextracellular signaling molecules convey their signals through theplasma membrane of the cell and thus elicit appropriate intracellularresponses.

Heterotrimeric G proteins are composed of three polypeptide subunits,namely G alpha, G beta and G gamma. The conformation the alpha subunitand the degree of association between all three subunits changes duringthe signal transduction process. These changes are associated with thehydrolysis of the nucleotide GTP to form GDP and Pi. The binding sitesfor GTP and GDP as well as the GTPase catalytic site reside in the alphasubunit.

The G protein cycle which occurs each time a signal is conveyed acrossthe membrane can be summarized as follows. In an unstimulated cell, theG proteins are found in the resting state in which alpha, beta and gammaare complexed together and GDP is bound to G alpha. The binding of anappropriate hormone or other signaling molecule to a G protein coupledreceptor at the cell surface initiates a signaling event when theligand-activated receptor stimulates the alpha subunit to exchange GDPfor GTP. In the active form, the alpha subunit, bound to GTP,dissociates from the beta-gamma complex, and the subunits then interactspecifically with cellular effector molecules to evoke a cellularresponse. G protein beta-gamma complexes, and in some instance alphasubunits, can interact with different effector systems (e.g.,phospholipase C, adenylyl cyclase systems, etc.) to evoke a variety ofcellular responses.

The present invention uses the ability of the dissociated beta-gammacomplex to specifically interact with cellular effector molecules toevoke a cellular response as an endpoint in assays to detectprotein-protein interactions. This ability of the beta-gamma complex tointeract with cellular effector molecules generally requires that thebeta-gamma complex be free of the alpha subunit. The present inventorshave discovered that interactions between the beta-gamma complexdissociated from the alpha subunit and an effector molecule can beprevented or inhibited by the association of the beta-gamma complex withanother unrelated protein.

The methods of the invention comprise the expression of a first proteinof interest fused in frame to a G protein gamma subunit. This G proteingamma subunit fusion is then expressed in the same cell as a second testprotein which is typically expressed in native form. Interactions, suchas binding, between the first protein fused to a G protein gamma subunitand the second test protein are assayed by determining whether the fusedG protein gamma subunit in its association with the beta subunit iscapable of interacting with effector molecules. In instances where thefirst protein fused to a G protein gamma subunit and the second proteininteract or bind, the fused G protein gamma subunit in its associationwith the beta subunit is not free to interact with effector molecules.In contrast, when the first protein fused to a G protein gamma subunitand the second protein do not interact or bind, the fused G proteingamma subunit in its association with the beta subunit is free tointeract with effector molecules (see FIG. 1).

Specific Embodiments

Definitions

As used herein, the term “agent” means any molecule that is randomlyselected or rationally designed. As used herein, an agent is said to berandomly selected when the agent is chosen randomly without consideringthe specific sequences involved in the association of the proteins understudy or the known functions of the proteins under study. Examples ofrandomly selected agents are chemical libraries, peptide combinatoriallibraries, or a growth broth of an organism.

As used herein, an agent is said to be rationally selected or designedwhen the agent is chosen on a nonrandom basis which takes into accountthe sequence of the proteins under study and/or their conformation inconnection with the agent's action. Agents can be rationally selected orrationally designed by utilizing the amino acid sequences that make uppotential contact sites between the proteins. For example, a rationallyselected peptide agent can be a peptide whose amino acid sequence isidentical to an identified contact site on one of the proteins understudy. Such an agent will reduce or block the association of the proteinwith its binding partner by binding to the contact site on the firstprotein.

The agents of the present invention can be, as examples, peptides, smallmolecules, nucleic acids, vitamin derivatives, as well as carbohydrates.A skilled artisan can readily recognize that there is no limit as to thestructural nature of the agents of the present invention.

The peptide agents of the invention can be prepared using standard solidphase (or solution phase) peptide synthesis methods, as is known in theart. In addition, the DNA encoding these peptides may be synthesizedusing commercially available oligonucleotide synthesis instrumentationand produced recombinantly using standard recombinant productionsystems. The production using solid phase peptide synthesis isnecessitated if non-gene-encoded amino acids are to be included.

Another class of agents are antibodies immunoreactive with one of theproteins under study. Particularly useful are antibodies immunoreactivewith the extracellular domain of membrane proteins under study. Asdescribed above, antibodies are obtained by immunization of suitablemammalian subjects with peptides, containing as antigenic regions, thoseportions of the protein intended to be targeted by the antibodies.Critical regions include the contact sites between the two proteins aswell as extracellular regions of membrane proteins.

As used herein, the phrase “at least one interaction” refers to aninteraction between the first protein fused to a G protein gamma subunitand the second protein detectable in the methods of the invention. Suchinteractions are typically, but not always, a form of direct interactionsuch as binding.

As used herein, the term “modulates” refers to any change in aninteraction between the first protein fused to a G protein gamma subunitand the second protein, wherein that change is detectable by the methodsof the invention. The term “modulates” includes inhibition or promotionof at least one interaction between a first protein fused to a G proteingamma subunit and the second protein.

As used herein, the term “cell” refers to any eukaryotic cell whichcontains a G protein coupled signaling system. Preferred cells include,but are not limited to, yeast cells such as Saccharomyces cerevisiae orSchizosaccharomyces pombe as well as insect, plant and mammalian cellsin culture.

As used herein, the term “fused” refers to a protein which has beenexpressed from a recombinant gene which comprises the coding sequencefrom the first protein covalently attached in frame to a G protein gammasubunit gene. This recombinant gene when transcribed and translatedproduces a chimeric protein comprising the amino acid sequence of thefirst protein and the amino acid sequence of a G protein gamma subunit.Typically, the first gene is fused in frame to the coding sequence of aG protein gamma subunit to produce a chimeric protein wherein the firstprotein is fused to the N or amino terminus of the G protein gammasubunit.

As used herein, the phrase “G protein gamma subunit” refers to anyavailable G protein gamma subunit from a eukaryotic cell. G proteingamma subunits are known to be highly variable with over eleven gammasubunit genes isolated and various splice variants identified. SeeHildebrand (1997) Biochem. Pharm. 54:325-339. The phrase also includesfragments of a G protein gamma subunit that retain the ability tointeract with a G protein beta subunit and retain the ability tointeract with at least one effector molecule. Preferably, G proteingamma subunits are selected from species corresponding to the selectedhost cell.

The term “first protein” refers to any protein employed in the methodsof the invention to detect protein-protein interactions. The first testprotein is fused to a G protein gamma subunit.

The term “second protein” refers to any protein employed in the methodsof the invention to detect protein-protein interactions. The second testprotein is not limited to any specific class of cellular proteins butspecifically includes membrane proteins.

As used herein, the term “effector molecule” refers to any G proteingamma-beta target that results in an assayable phenotype uponinteraction with a G protein gamma-beta complex. Many G protein effectormolecules or targets have been identified including ion channels I_(KG),and I_(Ca), phospholipase A₂, STE20, STE5, PLCβ, adenylyl cyclases, Gprotein coupled receptor kinases, P13K, the MAP kinase cascade, Brutontyrosine kinase, Tsk tyrosine kinase, arrestins and phosphodiesterases.See Clapham et al. ((1997) Ann. Rev. Pharmacol. Toxicol 37:167-203) fora review of known effector molecules or targets.

As used herein, the term “membrane protein” refers to a protein thatcomprises at least one membrane spanning domain. Such membrane spanningdomains typically comprise as series of hydrophobic amino acid residuesthat span the lipid bilayer membrane of a cell.

As used herein, the phrase “native form” refers to the expression of aprotein without alteration of its primary amino acid sequence and/orsecondary structure. For instance, a protein expressed in its nativeform is not expressed as a fusion protein.

As used herein, the term “sequestered” refers to any means of preventingthe interaction of a gamma-beta complex which is dissociated from thealpha subunit with an effector molecule. The term includes, but is notlimited to, the physical retention of the gamma-beta complex in or onthe cell membrane by the interaction of the first and second proteins.

As used herein, the term “library” generally refers to either acollection of cDNA molecules corresponding to a population of mRNAisolated from a selected cell type, or the proteins encoded by the cDNAlibrary. Methods to produce cDNA libraries from a selected cellpopulation are commonly available, such as the methods disclosed bySambrook et al. (Molecular Cloning, Cold Spring Harbor Laboratory Press,1989). cDNA libraries from selected cell types are also commerciallyavailable from vendors such as Stratagene®.

As used herein, the term “detectable phenotype” refers to any phenotypewhich may be detected by physical, visual or chemical means elicited bythe interaction of a beta-gamma complex with an effector molecule.Detectable phenotypes utilized in the invention include but are notlimited to yeast pheromone induced growth inhibition, other forms ofdetectable cellular morphology changes as well as the activities ofreporter genes such as lacZ and HIS3 operably linked to pheromoneresponsive promoters such as the BAR1 or FUS1 promoters (see U.S. Pat.No. 5,482,835).

Methods for detecting protein-protein interactions

In one embodiment, the present invention makes use of a gene hybrid thatencodes a first protein fused to a G protein gamma subunit.Interactions, such as binding, between the first protein fused to a Gprotein gamma subunit and the second test protein is assayed bydetermining whether the fused G protein gamma subunit in its associationwith the beta subunit is capable of interacting with effector moleculesupon induction of a G protein signaling cascade. In instances where thefirst protein fused to a G protein gamma subunit and the second testprotein interact or bind, the fused G protein gamma subunit in itsassociation with the beta subunit is not free to interact with effectormolecules. In contrast, when the first protein fused to a G proteingamma subunit and the second protein do not interact or bind, the fusedG protein gamma subunit in its association with the beta subunit is freeto interact with effector molecules (see FIG. 1).

The methods of the invention include providing a host cell, preferably ayeast cell, most preferably Saccharomyces cerevisiae orSchizosaccharomyces pombe. In one format, a first chimeric gene isprovided. The first chimeric gene comprises a DNA sequence that encodesa first protein or fragment fused to a G protein gamma subunit.

A second gene is provided which is capable of being expressed in thehost cell. The second gene encodes a second test protein which willtypically be expressed in its native form. In one embodiment, both thefirst and the second genes are introduced into the host cell cloned intoappropriate plasmids or expression vectors. The first chimeric gene mayalso be present in a chromosome of the host cell and the second gene isintroduced into the host cell as part of a vector, or vice-versa. Inanother embodiment, the second gene may be natively expressed in thehost cell.

Vectors useful for practicing the present invention include plasmids,viruses, and integratable DNA fragments (i.e., fragments integratableinto the host genome by homologous or nonhomologous recombination). Thevector may replicate and function independently of the host genome, asin the case of a plasmid, or may integrate into the genome itself, as inthe case of an integratable DNA fragment. Suitable vectors will containreplicon and control sequences which are derived from species compatiblewith the intended expression host. For example, a promoter operable in ahost cell is one which binds the RNA polymerase of that cell, and aribosomal binding site operable in a host cell is one which binds theendogenous ribosomes of that cell.

Transformation or transfection of the vectors into suitable host cellsmay be accomplished by any means available, such as those disclosed byAusubel et al. ((1987), Current Protocols in Molecular Biology,Wiley-Interscience, New York.). Transformed host cells employed in thepresent invention are cells which have been transformed or transfectedwith the vectors constructed using recombinant DNA techniques andexpress the protein or protein subunit coded for by the heterologous DNAsequences. The host cells may be, but are not required to be, incapableof expressing an endogenous G protein gamma-subunit corresponding to theG protein gamma subunit to which the first protein is fused.

Although the methods of the present invention are not limited to yeastcells, a variety of yeast cultures, and suitable expression vectors fortransforming yeast cells, are known. See, e.g., U.S. Pat. Nos.4,745,057; 4,797,359; 4,615,974; 4,880,734; 4,711,844; and 4,865,989.Saccharomyces cerevisiae is the most commonly used among the yeast,although a number of other strains are commonly available. See, e.g.,U.S. Pat. No. 4,806,472 (Kluveromyces lactis and expression vectorstherefor); and U.S. Pat. No. 4,855,231 (Pichia pastoris and expressionvectors therefor).

Yeast vectors may contain an origin of replication from the 2 micronyeast plasmid or an autonomously replicating sequence (ARS), a promoter,DNA encoding the heterologous DNA sequences, sequences forpolyadenylation and transcription termination, and a selection gene. Anexemplary plasmid is YRp7, (Stinchcomb et al., (1979) Nature 282:39;Kingsman et al., (1979) Gene 7:141; or Tschemper et al., (1980) Gene10:157). This plasmid contains the TRP1 gene, which provides a selectionmarker for a mutant strain of yeast lacking the ability to grow intryptophan, for example ATCC No. 44076 or PEP4-1 (Jones, (1977) Genetics85, 12). The presence of the trp1 lesion in the yeast host cell genomethen provides an effective environment for detecting transformation bygrowth in the absence of tryptophan.

Suitable promoting sequences in yeast vectors include the promoters formetallothionein, 3-phosphoglycerate kinase (Hitzeman et al., (1980) J.Biol. Chem. 255: 2073 or other glycolytic enzymes (Hess et al., (1968) JAdv. Enzyme Reg. 7:149; and Holland et al., (1978) Biochemistry17:4900), such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase. Suitable vectors and promoters for use in yeast expressionare further described in R. Hitzeman et al., EPO Publn. No. 73,657.Other promoters, which have the additional advantage of transcriptioncontrolled by growth conditions, are the promoter regions for alcoholdehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymesassociated with nitrogen metabolism, and the aforementionedmetallothionein and glyceraldehyde-3-phosphate dehydrogenase, as well asenzymes responsible for maltose and galactose utilization.

In constructing suitable expression plasmids, the termination sequencesassociated with these genes may also be ligated into the expressionvector 3′ of the heterologous coding sequences to providepolyadenylation and termination of the mRNA.

As discussed above, induction of a G protein signaling cascade may berequired to induce a detectable phenotype which is dependent upon theinteraction of the first and second proteins used in the invention. Thechoice of inducing agent depends in part upon the choice of detectablephenotype selected as well as the effector molecule involved in thesignaling pathway. For instance, in yeast wherein pheromone inducedgrowth inhibition is used as the detectable phenotype, α-factorpheromone is employed to induce the G protein signaling cascade.Alteratively, available agonists of any G protein-coupled receptor maybe used to induce a specific phenotype such as hormones,neurotransmitters and growth factors (see Muller et al. (1995) Biochem.Soc. Trans. 23(1):141-8 and Dohlman et al. (1997) J. Biol. Chem.272(7):3871-3874). Exposure to differing amounts of an inducing agentmay be used to modulate the detectable phenotype.

The above methods may be modified to identify agents which modulate atleast one interaction between the first and second proteins. In thisformat, vectors encoding a first protein fused to a G protein gammasubunit and a second protein are prepared. Generally, the first andsecond protein will be previously identified. Further, the interactionbetween the first protein and the second protein will typically beassociated with a given disease state, condition or clinical indication.

Cells which express a first protein fused to a G protein gamma subunitand a second protein are exposed or incubated with the agent. The cellsare then assayed for the ability of the agent to modulate an interactionbetween the first and second protein. In this format, cells exposed tothe agent which inhibits an interaction between the first and secondproteins will exhibit changes in the detectable phenotype being assayed.This change in detectable phenotype occurs, after induction of a Gprotein signaling cascade, when the G protein subunits dissociate and Gbeta/gamma is free to activate a signaling pathway leading to thedetectable phenotype. In control cells not exposed to the agent, theinteraction of the first and second proteins prevents beta/gamma frominteracting with the normal effector to activate the signaling pathway.

The above methods may be used to identify new, previously unidentifiedbinding partners for a known protein. In one format, a known protein maybe expressed in its native form as the second protein to screen forpotential binding partners expressed from a library of G protein gammasubunit fusions as the first protein. In another format, the firstprotein may be a known protein expressed as a G protein gamma subunitfusion to screen for potential binding partners expressed as the secondprotein from a cDNA library.

The methods of the invention may also be modified to detect and identifymutations which modulate protein-protein interactions between twobinding partners. As an example, mutations in a first protein fused to aG protein gamma subunit which affect the binding to a second protein maybe assayed by preparing a library of mutations fused in frame to the Gprotein gamma subunit. Mutations may be produced by any means availablein the art, such as site directed (oligonucleotide-directed)mutagenesis, linker insertion or scanning mutagenesis, deletionmutagenesis or mutations induced in Sec-1 by chemical mutagenesis. SeeSambrook et al. for a variety of available site-directed and randommutagenesis methods.

In an alternative format, mutations in a second protein which affect thebinding of the second protein to a first protein fused to a G proteingamma subunit may be assayed by preparing a library of mutations in thesecond protein. Mutations in the second protein may also be preparedusing any available means including site directed(oligonucleotide-directed) mutagenesis, linker insertion or scanningmutagenesis, deletion mutagenesis or mutations induced by chemicalmutagenesis.

The methods of the present invention, as described above, may bepracticed using a kit for detecting interactions between a first testprotein and a second test protein. The kit may include a container, twovectors, and a host cell. The first vector contains a promoter and mayinclude a transcription termination signal functionally associated withthe coding sequence for a G protein gamma subunit. The first vectorincludes a restriction site(s) for inserting a DNA sequence encoding afirst test protein or protein fragment in such a manner that the firsttest protein is expressed as part of a fusion protein with the G proteingamma subunit. The first vector may include the coding sequence for aknown protein fused in frame to the coding sequence for a G proteingamma subunit. Alternatively, the first vector may be supplied as apopulation of vectors comprising a library of cDNA fragments fused inframe to the coding sequence for a G protein gamma subunit.

The kit includes a second vector which includes a promoter and atranscription termination signal to direct transcription. The secondvector also includes a restriction site(s) to insert a DNA sequenceencoding the second test protein or protein fragment into the vector, insuch a manner that the second test protein is capable of being expressedin a relevant host cell. The second vector may include the codingsequence for a known protein. Alternatively, the second vector may besupplied as a population of vectors comprising a library of cDNAfragments.

The kit may also include a host cell, preferably a yeast strain ofSaccharomyces cerevisiae or Schizosaccharomyces pombe.

The following working examples specifically point out preferredembodiments of the present invention, and are not to be construed aslimiting in any way the remainder of the disclosure. Other genericconfigurations will be apparent to one skilled in the art.

EXAMPLE 1 Production of G Protein Gamma Subunit Fusion Vectors.

The production of G protein gamma fusions to a protein of interest maybe accomplished using any available vectors designed to replicate andexpress the fusion protein in the appropriate host cell.

When yeast cells are used as the host cell, pRS316-GAL-STE4 wasconstructed by ligation of the EcoRI-SalI fragment from pL19, containingthe bidirectional galactose-inducible GAL1/10 promoter and STE4 (Gβgene) (Whiteway et al., (1990) Overexpression of the STE4 gene leads tomating response in haploid Saccharomyces cerevisiae, Molecular andCellular Biology 10:217-222.) into the vector pRS316 (described bySikorski et al., (1989) A system of shuttle vectors and yeast hoststrains designed for efficient manipulation of DNA in Saccharomycescerevisiae. Genetics 122:19-27.). STE18 (Gγ gene) was PCR amplifiedusing the plasmid M57p4 as the template (Whiteway et al.) andoligonucleotides that flank the STE18 open reading frame (GGG AAT TCAATG GCT CAC CAT CAC CAT CAC CAT GCT AGC ATG ACA TCA GTT CAA AAC TCT CC,(SEQ ID NO: 1) 5′ oligo, encoding 6 His residues upstream of the aminoterminus; GAG GCT CTA CGT AGC AAG, (SEQ ID NO: 2) 3′ oligo). The PCRproduct was digested with EcoRI and SacI, and ligated to thecorresponding sites in pRS316-GAL5 STE4 described above to producepRS316-GAL-STE4/18-H6.

pRS314-GAL-H6-STE18 (FIG. 2) was constructed by ligation of theBamHI-SacI fragment from pRS316-GAL-STE4/18-H6 into pRS314. To preparefusions of a protein of interest to the yeast G protein gamma subunit,the coding sequence of the protein is inserted in frame into the EcoRIsite of pRS314-GAL-H6-STE18 to yield pRS314-GAL-heterologousgene-H6-STE18.

EXAMPLE 2 A Method of Detecting the Interaction Between Two Proteins

Production of a Rat Sec-1-G Protein Gamma Subunit Fusion andTransformation of host Cells Expressing the Sec-1 G Protein GammaSubunit Fusion and Syntaxin.

Strains, Media and Transformation:

Standard methods for the growth, maintenance, and transformation ofyeast and bacteria, and for the manipulation of DNA, were usedthroughout (Ausubel et al. 1987, Current Protocols in Molecular Biology.Wiley-Interscience, New York). The Escherichia coli strain DH5α was usedfor the maintenance of plasmids. Yeast cells were grown in syntheticmedium supplemented with amino acids, adenine and 2% glucose (SCD) or 2%galactose plus 0.2% sucrose (SCG); tryptophan and/or uracil were omittedto maintain selection for plasmids. The Saccharomyces cerevisiae strainused in this study was MHY6 (from Jeremy Thorner) (MATa ura3-52lys2-801^(am) ade2-101^(oc) trp1-Δ63 his3-Δ200 leu2-Δ1 ste18Δ::LEU2)(derived from Sikorski et al. (1989) Genetics 122(1):19-27).

Plasmid Construction:

pRS316-GAL-STE4 was constructed by ligation of the EcoRI-SalI fragmentfrom pL19, containing the bidirectional galactose-inducible GAL1/10promoter and STE4 (Gβ gene) (from Whiteway et al.) into the vectorpRS316 (described in Sikorski et al., (1989) A system of shuttle vectorsand yeast host strains designed for efficient manipulation of DNA inSaccharomyces cerevisiae. Genetics 122:19-27.). STE18 (Gγ gene) was PCRamplified using the plasmid M57p4 as the template (from Whiteway et al.)and oligonucleotides that flank the STE18 open reading frame (GGG AATTCA ATG GCT CAC CAT CAC CAT CAC CAT GCT AGC ATG ACA TCA GTT CAA AAC TCTCC, (SEQ ID NO: 1) 5′ oligo, encoding 6 His residues upstream of theamino terminus; GAG GCT CTA CGT AGC AAG, (SEQ ID NO: 2) 3′oligo). ThePCR product was digested with EcoRI and SacI, and ligated to thecorresponding sites in pRS316-GAL-STE4 described above to producepRS316-GAL-STE4/18-H6.

pRS314-GAL4-H6-STE18 was constructed by ligation of the BamHI-SacIfragment from pRS316-GAL-STE4/18-H6 into pRS314. Rat brain Sec1 (rbSec1)was PCR amplified using a plasmid that contains the rbSEC cDNA inpBluescript II SK as a template (Garcia et al., (1994) A rat brain Sec1homologue related to Rop and UNC18 interacts with syntaxin. Proc. Nat'lAcad. Sci. USA 91:2003-7) and oligonucleotides that flank the Sec1 openreading frame (CCG AAT TCC ATG GCC ATT GGC CTC AAG, (SEQ ID NO:3) 5′oligo, containing an EcoRI site beginning 7 nucleotides upstream of theinitial ATG; CCT GAA TTC GAG CTT ATT TCT TCG TCT GTT TTA TTC AG, (SEQ IDNO: 4) 3′ oligo, containing an EcoRI site in frame with the N-terminalopen reading frame of His6-STE18). The rbSec1 PCR product was digestedwith EcoRI and ligated into the EcoRI site of pRS314-GAL-H6-STE18 toyield pRS314-GAL-rbSec1-H6-STE18.

pRS316-ADH (amp^(r), CEN/ARS, URA3 and ADH1 promoter and terminationsequence) (described in Song et al., (1996) Regulation of membrane andsubunit interactions by N-myristoylation of a G protein α subunit inyeast. J. Biol. Chem. 271:20273-20283.). Rat syntaxin 1a was PCRamplified using a cDNA in vector CDM8 as the template, (Blasi et al.(1993). Botulinum neurotoxin C1 blocks neurotransmitter release by meansof cleaving HPC-1/syntaxin. EMBO J. 12:4821-8.) and oligonucleotidesthat flank the syntaxin open reading frame (CGC GGG TCG ACA TGA AGG ACCGAA CCC AGG, (SEQ ID NO:5) 5′ oligo, containing a SalI site beginning 6nucleotides upstream of the initial ATG; CCG AAT TCA CTA TCC AAA GAT GCCCCC GAT GG, (SEQ ID NO: 6) 3′ oligo, containing an EcoRI site 10nucleotides downstream of the termination codon). The PCR amplifiedproduct was digested with SalI and EcoR1 and ligated to thecorresponding sites in pRS316-ADH.

Detection of Sec-1-Syntaxin Interactions

Growth Inhibition Assay:

To evaluate to the interactions of the Sec-1 G protein gamma subunitfusion and syntaxin, a growth inhibition assay (halo assay) was carriedout by diluting 50 μL from an overnight culture grown at 30° C. inselective media with 2 mL sterile water, followed by the addition of anequal volume of 1% (w/v) dissolved agar (55° C.) and poured onto aculture dish of SCD-Trp-Ura or SCG-Trp-Ura medium. Sterile filter discswere spotted with 5 μg or 15 μg of synthetic α-factor pheromone andplaced onto the nascent lawn to induce growth arrest. The resulting zoneof growth-arrested cells was documented after 2 days at 30° C.

As set forth in FIG. 3, yeast expressing the Sec-1 G protein gammasubunit fusion and syntaxin did not respond to α-factor induced growtharrest as exhibited by no zone of inhibition around filter paper diskscontaining 5 or 15 μg of α-factor. This is in contrast to control cellswhich express the Sec-1 G protein gamma fusion alone and cells whichexpress syntaxin alone. In the control cells, upon pheromone addition,the G protein subunits dissociate and G beta/gamma is free to activate asignaling pathway leading to growth arrest. When the cells express theSec-1 G protein gamma fusion and syntaxin, pheromone induced growtharrest is blocked because the interaction of syntaxin with Sec1 preventsthe beta/gamma from interacting with the normal effector to activate thesignaling pathway to growth arrest.

EXAMPLE 3 Methods for Testing the Ability of an Agent to Modulate anInteraction Between Two Proteins

The system described in Example 2 may be used to screen for agents whichmodulate the interactions between any two proteins. As an example,agents which modulate the interactions between Sec-1 and syntaxin may bescreened by exposing yeast cells expressing the Sec-1 g protein gammafusion and syntaxin to the agent to be tested. A growth inhibition assay(halo assay) is carried out by diluting 50 μL from an overnight cultureof yeast expressing the Sec-1 G protein gamma fusion and syntaxin at 30°C. in selective media with 2 mL sterile water, followed by the additionof an equal volume of 1% (w/v) dissolved agar (55° C.) and poured onto aculture dish of SCD-Trp-Ura or SCG-Trp-Ura medium with and without theagent to be tested. Sterile filter discs are spotted with 5 μg or 15 μgof synthetic α-factor pheromone and placed onto the nascent lawn toinduce growth arrest. Alternatively, the agent to be tested may bespotted onto filter disks with the synthetic α-factor pheromone. Theresulting zone of growth-arrested cells is then documented after 2 daysat 30° C.

In control cells not exposed to the agent, pheromone induced growtharrest is blocked because the interaction of syntaxin with Sec1 preventsbeta/gamma from interacting with the normal effector to activate thesignaling pathway to growth arrest. In cells exposed to an agent whichinhibits the interaction of Sec-1 and syntaxin, upon pheromone addition,the G protein subunits dissociate and G beta/gamma is free to activate asignaling pathway leading to growth arrest. This pheromone inducedgrowth arrest is exhibited by a zone of inhibition around the filterdisk.

In a second assay, agents which modulate the interactions between theHer-2/neu receptor tyrosine kinase which is upregulated in somecancerous cells, including some breast cancers, and Grb2 may be screenedby exposing yeast cells expressing a Grb2-G protein gamma fusion andHer-2/neu to the agent to be tested. A vector comprising a Grb2-Gprotein gamma fusion is prepared by inserting the coding sequence forGrb2 (available in Lowenstein et al. (1992) Cell 70(3):431--442) intopRS314-GAL4-H6-STE18. A second vector encoding-Her-2/neu is prepared byinsertion of the coding sequence of Her-2/neu (available in Yamamoto etal (1986) Nature 319:230-234) into pRS316-ADH.

A growth inhibition assay (halo assay) is carried out by diluting 50 μLfrom an overnight culture of yeast expressing the Grb2-G protein gammafusion and Her-2/neu at 30° C. in selective media with 2 mL sterilewater, followed by the addition of an equal volume of 1% (w/v) dissolvedagar (55° C.) and poured onto a culture dish of SCD-Trp-Ura orSCG-Trp-Ura medium with and without the agent to be tested. Sterilefilter discs are spotted with 5 μg or 15 μg of synthetic α-factorpheromone and placed onto the nascent lawn to induce growth arrest.Alternatively, the agent to be tested may be spotted onto filter diskswith the synthetic α-factor pheromone. The resulting zone ofgrowth-arrested cells is then documented after 2 days at 30° C.

In control cells not exposed to the agent pheromone induced growtharrest is blocked because the interaction of activated Her-2/neu withGrb2 prevents beta/gamma from interacting with the normal effector toactivate the signaling pathway to growth arrest. In cells exposed to anagent which inhibits the interaction of activated Her-2/neu and Grb2,upon pheromone addition, the G protein subunits dissociate and Gbeta/gamma is free to activate a signaling pathway leading to growtharrest. This pheromone induced growth arrest is exhibited by a zone ofinhibition around the filter disk.

EXAMPLE 4 Method for Identifying Binding Partners for a Protein

The system described in Example 2 may also be used to identify new,previously unidentified binding partners for a known protein. In oneformat, a known protein may be expressed in its native form, such assyntaxin, to screen for potential binding partners expressed from alibrary of G protein gamma subunit fusions. Syntaxin is a known targetfor botulinum toxin. Botulinum toxin is currently used as a therapeuticagent to treat central nervous system disorders such as spasticity amongstroke patients. The identification of new binding partners for syntaxinmay elucidate alternate potential drug targets for the treatment of suchdisorders.

Rat syntaxin 1a is PCR amplified using a cDNA in vector CDM8 as thetemplate, (Blasi, J., E. R. Chapman, S. Yamasaki, T. Binz, H. Niemann,and R. Jahn, 1993. Botulinum neurotoxin C1 blocks neurotransmitterrelease by means of cleaving HPC-1/syntaxin. EMBO Journal 12:4821-8) andoligonucleotides that flank the syntaxin open reading frame (CGC GGG TCGACA TGA AGG ACC GAA CCC AGG, (SEQ ID NO: 5) 5′ oligo, containing a SalIsite beginning 6 nucleotides upstream of the initial ATG; CCG AAT TCACTA TCC AAA GAT GCC CCC GAT GG, (SEQ ID NO: 6) 3′ oligo, containing anEcoRI site 10 nucleotides downstream of the termination codon). The PCRamplified product was digested with SalI and EcoRI and ligated to thecorresponding sites in pRS316-ADH to facilitate expression of ratsyntaxin 1a in a recombinant yeast cell.

First Protein Library Fusions to G Protein gamma Subunit

A library of G Protein gamma subunit fusions is prepared by ligating acDNA library of choice into the EcoRI site of pRS314GAL-H6-STE18 asdescribed above. Alternatively, appropriate vector backbones may besubstituted depending on the cell in which the G protein gamma subunitfusions are to be expressed.

To prepare the library of G protein gamma subunit fusions, the cDNAinserts from a rat brain cDNA library purchased from Stratagene (Cat.No. 936501) are released from the backbone vector using standardprocedures and inserted into the EcoRI site of pRS314-GAL-H6-STE18. Thislibrary is then transformed into yeast which express rat syntaxin 1a anda lacZ reporter gene fused downstream from the FUS1 pheromone induciblepromoter (Nomoto et al. (1990) EMBO J. 9:691) by the methods of Ausubelet al.

Screening and identification for proteins which interact with syntaxinare accomplished by assaying for the ability of individual fusionproteins from the G protein gamma subunit fusion library to interactwith syntaxin after pheromone induction. In cells which express a Gprotein gamma subunit fusion which does not interact with syntaxin, uponpheromone exposure, the G protein subunits dissociate and G beta/gammais free to activate a signaling pathway leading to transcription of thelacZ reporter gene fused downstream from the FUS1 promoter. In cellswhich express a G protein gamma fusion which interacts with syntaxin,pheromone induced transcription from the lacZ reporter gene fuseddownstream from the FUS1 promoter is blocked. In this case, theinteraction of the G protein gamma subunit fusion with syntaxin preventsbeta-gamma from activating a signaling pathway leading to transcriptionof the lacZ reporter gene fused downstream from the FUS1 promoter

Individual G protein gamma subunit fusions which may interact withsyntaxin are isolated by recovery and sequencing of the cDNA insertfused to the coding sequence for the G protein gamma subunit by standardcloning and sequencing techniques. Such techniques are widely availablesuch as those disclosed by Sambrook et al. (Molecular Cloning: ALaboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press,Plainview N.Y., 1989).

Expression of cDNA Libraries as the Second Protein.

In an alternative format, the rat brain library may be screened forproteins which interact with the Sec-1/G protein gamma subunit fusionexpressed by pRS314-GAL-rbSec1-H6-STE18. In this format, he rat brainlibrary cDNA described above is released from the backbone vector usingstandard procedures and inserted into pRS316-ADH. This library is thenco-transformed into cells which contain a lacZ reporter gene fuseddownstream from the FUS1 pheromone inducible promoter (Nomoto et al.(1990) EMBO J. 9:691) with pRS314-GAL-rbSec1-H6-STE18.

Proteins which interact with Sec-1 are identified by assaying for theability of individual proteins encoded by the library to interact withSec-1 after pheromone induction. In cells which express a proteinencoded by a member of the rat cDNA library which does not interact withthe Sec-1/G protein gamma subunit fusion, upon pheromone exposure, the Gprotein subunits dissociate and G beta-gamma is flee to activate asignaling pathway leading to transcription of the lacZ reporter genefused downstream from the FUS1 promoter. In cells which express alibrary protein which does interact with the Sec-1/G protein gammafusion, pheromone induced transcription from the lacZ reporter genefused downstream from the FUS1 pheromone inducible promoter is blocked.In this case, the interaction of the Sec-1/G protein gamma subunitfusion with the library protein prevents the beta/gamma from activatinga signaling pathway leading to transcription of the lacZ reporter genefused downstream from the FUS1 promoter.

Individual cDNAs encoding library proteins which may interact with Sec-1are isolated by recovery and sequencing of the cDNA insert by standardcloning and sequencing techniques. Such techniques are widely availablesuch as those disclosed by Sambrook et al. (Molecular Cloning: ALaboratory Manual, 2nd edition, Cold SPRING Harbor Laboratory Press,Plainview N.Y., 1989).

EXAMPLE 5 Method for Identifying Mutations which Disrupt Protein-ProteinInteractions.

The system described in Example 2 may also be used to screen for theeffects of mutations which modulate protein-protein interactions betweentwo binding partners. As an example, mutations in Sec-1 which effect thebinding of Sec-1 to syntaxin may be assayed by preparing a library ofSec-1 mutations fused in frame to the G protein gamma subunit asdescribed above. Mutations in Sec-1 may be produced by any meansavailable in the art such as site directed (oligonucleotide-directed)mutagenesis, linker insertion or scanning mutagenesis, deletionmutagenesis or mutations induced in Sec-1 by chemical mutagenesis. SeeSambrook et al. for a variety of available site-directed and randommutagenesis methods.

Mutations in Sec-1 are then screened for their ability to disrupt Sec-1binding to syntaxin by transforming cells which express syntaxin frompRS316-ADH-syntaxin with pRS314-GAL-rbSec1(mut)-H6-STE18 (rbSec-1(mut)encoding a Sec-1 mutation). Growth inhibition assays (halo assays) arecarried out by diluting 50 μL from an overnight culture grown at 30° C.in selective media with 2 mL sterile water, followed by the addition ofan equal volume of 1% (w/v) dissolved agar (55° C.) and poured onto aculture dish of SCD-Trp-Ura or SCG-Trp-Ura medium. Sterile filter discsare spotted with 5 μg or 15 μg of synthetic α-factor pheromone andplaced onto the nascent lawn to induce growth arrest. The resulting zoneof growth-arrested cells is documented after 2 days at 30° C.

Yeast expressing wild-type (non-mutated) Sec-1 G protein gamma subunitfusion and syntaxin do not respond to α-factor induced growth arrest asexhibited by no zone of inhibition around filter paper disks containing5 or 15 μg of α-factor. This is contrast to cells which express Sec-1 Gprotein gamma fusions wherein Sec-1contains a mutation that disruptsSec-1-syntaxin binding. In these cells, upon pheromone addition, the Gprotein subunits dissociate and G beta/gamma is free to activate asignaling pathway leading to growth arrest.

In an alternative format, mutations in syntaxin which affect the bindingof syntaxin to Sec-1 may be assayed by preparing a library of syntaxinmutations as described above. Mutations in syntaxin may be prepared byany available means including site directed (oligonucleotide-directed)mutagenesis, linker insertion or scanning mutagenesis, deletionmutagenesis or mutations induced in syntaxin by chemical mutagenesis.

Mutations in syntaxin are then screened for their ability to disruptsyntaxin binding to Sec-1 by transforming cells which express a Sec-1Gprotein gamma subunit fusion, such as that encoded bypRS314-GAL-rbSec1-H6-SYE18 with pRS316 ADH-syntaxin* (wherein“syntaxin*” refers to a syntaxin mutation). Growth inhibition assays(halo assays) are carried out by diluting 50 μL from an overnightculture grown at 30° C. in selective media with 2 mL sterile water,followed by the addition of an equal volume of 1% (w/v) dissolved agar(55° C.) and poured onto a culture dish of SCD-Trp-Ura or SCG-Trp-Uramedium. Sterile filter discs are spotted with 5 μg or 15 μg of syntheticα-factor pheromone and placed onto the nascent lawn to induce growtharrest. The resulting zone of growth-arrested cells is documented after2 days at 30° C.

Yeast expressing the Sec-1 G protein gamma subunit fusion and wild-type(non-mutated) syntaxin do not respond to α-factor induced growth arrestas exhibited by the absence of a zone of inhibition around filter paperdisks containing 5 or 15 μg of α-factor. This is contrast to cells whichexpress the Sec-1 G protein gamma subunit fusion and syntaxin whichcontains a mutation that disrupts syntaxin-Sec-1 binding. In thesecells, upon pheromone addition, the G protein subunits dissociate and Gbeta/gamma is free to activate a signaling pathway leading to growtharrest.

It should be understood that the foregoing discussion and examplesmerely present a detailed description of certain preferred embodiments.It therefore should be apparent to those of ordinary skill in the artthat various modifications and equivalents can be made without departingfrom the spirit and scope of the invention. Once such variation includesthe use of other signaling cascades which transmit signal throughmembrane proteins into the cell. All references, articles and patentsidentified above are herein incorporated by reference in their entirety.

6 1 62 DNA Artificial Sequence Description of Artificial Sequence 5′ PCRprimer for STE18 (G gamma gene) 1 gggaattcaa tggctcacca tcaccatcaccatgctagca tgacatcagt tcaaaactct 60 cc 62 2 18 DNA Artificial SequenceDescription of Artificial Sequence 3′ PCR primer for STE18 (G gammagene) 2 gaggctctac gtagcaag 18 3 27 DNA Artificial Sequence Descriptionof Artificial Sequence 5′ cloning primer for GAL4-STE18 sequence 3ccgaattcca tggccattgg cctcaag 27 4 38 DNA Artificial SequenceDescription of Artificial Sequence 3′ cloning primer for GAL4-STE18sequence 4 cctgaattcg agcttatttc ttcgtctgtt ttattcag 38 5 30 DNAArtificial Sequence Description of Artificial Sequence 5′ PCR primer forrat syntaxin 1a gene 5 cgcgggtcga catgaaggac cgaacccagg 30 6 32 DNAArtificial Sequence Description of Artificial Sequence 3′ PCR primer forrat syntaxin 1a gene 6 ccgaattcac tatccaaaga tgcccccgat gg 32

What is claimed is:
 1. A method for detecting one or more interactionsbetween two proteins, comprising the steps of: (a) providing a cell witha first protein, which is not a heterotrimeric G protein or subunitthereof, fused to a G protein gamma subunit and providing the same cellwith a second protein; and (b) determining whether the interaction ofthe G protein gamma subunit with an effector molecule has beenmodulated, thereby determining whether the first and second proteinsinteract.
 2. The method of claim 1, wherein said cell does not comprisean endogenous G protein gamma subunit capable of interacting with theeffector molecule.
 3. The method of claim 1, wherein the cell doesexpress an active endogenous effector molecule.
 4. The method of claim1, wherein the first protein is an unknown member of a library ofproteins fused to a G protein gamma subunit.
 5. The method of claim 1,wherein the second protein is a membrane protein.
 6. The method of claim1, wherein the effector molecule is selected from the group consistingof kinases, arrestins, nucleotide cyclases, phosphodiesterases,phospholipase, small molecule transporters and channels.
 7. The methodof claim 1, wherein the second protein is expressed in its native form.8. The method of claim 1, wherein the interaction of the G protein gammasubunit with an effector molecule confers a detectable phenotype uponthe host cell.
 9. The method of claim 1, wherein the second protein isan unknown protein encoded by a cDNA library.
 10. A method ofidentifying binding partners of a protein, comprising the steps of: (a)transforming or transfecting host cells with a library comprising apopulation of nucleic acid molecules which express a first protein,which is not a heterotrimeric G protein or subunit thereof, fused to a Gprotein gamma subunit to produce a host cell population, said populationof nucleic acids differing with respect to the first protein fused to aG protein gamma subunit; (b) transforming the host cell population witha vector which expresses a second protein; (c) culturing said host cellunder conditions which express said first and second protein; and (d)determining the activity of an effector molecule which interacts withthe G protein gamma subunit.
 11. A method of identifying bindingpartners of a protein, comprising the steps of: (a) transforming ortransfecting host cell with a nucleic acid molecule which expresses afirst protein, which is not a heterotrimeric G protein or subunitthereof, fused to a G protein gamma subunit; (b) transforming the hostcell with a vector which expresses a second protein; (c) culturing saidhost cell under conditions which express said first and second protein;and (d) determining the activity of an effector molecule which interactswith the G protein gamma subunit.
 12. The method of claim 11, whereinthe second protein is an unknown protein encoded by a cDNA library. 13.A method for identifying mutations in a protein which effect one or moreinteractions between two proteins, comprising the steps of: (a)providing a cell with a first protein, which is not a heterotrimeric Gprotein or subunit thereof, fused to a G protein gamma subunit and asecond protein presumed to contain the mutation; and (b) determiningwhether the interaction of the G protein gamma subunit with an effectormolecule has been modulated, thereby determining whether theinteractions of the first and second proteins is effected by themutation.
 14. A method of identifying mutations in a protein whichaffect one or more interactions between two proteins, comprising thesteps of: (a) transforming or transfecting a host cell with a nucleicacid molecule which expresses a first protein, which is not aheterotrimeric G protein or subunit thereof, fused to a G protein gammasubunit; (b) transforming the host cell with a vector which expresses asecond protein; (c) culturing said host cell under conditions whichexpress said first and second protein; and (d) determining the activityof an effector molecule which interacts with the G protein gammasubunit.
 15. The method of claim 12, wherein either the first or secondproteins contain a mutation.